Dual blockade of P-selectin and beta2-integrin in the liver inflammatory response after uncontrolled hemorrhagic shock.

BACKGROUND: Neutrophil infiltration is a characteristic feature of the hepatic injury associated with prolonged hypotension. Previous work has already stressed the important contribution of neutrophil-endothelial cell interactions in the organ injury seen after hemorrhagic shock. Single-blockade strategies using monoclonal antibodies (MAbs) against either selectin or integrin receptors have been demonstrated to be effective in limiting the tissue inflammatory response observed in this clinical disorder. One unexplored topic is the additive effect(s) and the potential antiinflammatory properties of the combined blocking of P-selectin plus beta2-integrin in the liver inflammatory response after uncontrolled hemorrhagic shock in rats. STUDY DESIGN: Sprague-Dawley rats (n = 64) weighing 250-300 g were included in a three-phase model of uncontrolled hemorrhagic shock. A prehospital phase consisted of 90 minutes of fluid resuscitation with lactated Ringer's solution to reach a mean arterial pressure (MAP) of 40 mmHg; a hospital phase consisted of 60 minutes of hemostasis and fluid resuscitation with lactated Ringer's solution to reach a MAP of 80 mmHg; and the third phase was 3 days of observation. All rats had 3 mL/100 g of blood volume shed during the initial 15 minutes. At 30 minutes, 75% tail amputation produced uncontrolled hemorrhagic shock. Four groups were randomized (n = 16 per group), and treatment at the beginning of resuscitation included normal saline (group 1); anti-P-selectin MAb, RMP-1 (group 2); anti-beta2-integrin MAb, WT.3 (group 3); or anti-P-selectin plus anti-beta2-integrin MAbs (group 4). The following indices were evaluated: fluid requirements for resuscitation, liver injury tests, liver tissue myeloperoxidase, and liver histology. RESULTS: Dual blockade of P-selectin and beta2-integrin significantly reduced fluid requirements for resuscitation (p < 0.05). We also observed a statistically significant improvement (p < 0.05) in tests demonstrating hepatic injury, myeloperoxidase in hepatic tissue, and histology studies. Survival was increased from 40% in controls to 60% with the dual-blockade treatment. CONCLUSIONS: These results indicate that dual-blockade strategies aimed at P-selectin and beta-integrin provided a protective effect in the liver inflammatory response after uncontrolled hemorrhagic shock in rats. Although dual blockade was more effective than either individual blockade alone, questions remain about the possible redundancy in the inflammatory adhesion pathways after this clinical condition.

Sialyl Lewis(x) analog improves liver function by decreasing neutrophil migration after hemorrhagic shock.

BACKGROUND: Little is known about the changes in the hepatic microcirculation and the leukocyte-endothelial adhesion processes during the early reperfusion period after resuscitation in hemorrhagic shock. P-selectin and its natural ligand Sialyl Lewis(x) (SLe(x)) are involved in the early stages of reperfusion events leading to neutrophil migration. Therefore, the aim of this study was to investigate the effect of the administration of CY-1503 [corrected], a synthetic SLe(x) analog, in the liver inflammatory response and neutrophil migration after hemorrhagic shock. MATERIALS AND METHODS: Rats, each weighing 275 to 300 grams, were subjected to 60 minutes of pressure controlled hemorrhagic shock. After this period, animals were resuscitated according to the following protocol: shed blood was reinfused to equal 50% of the total volume bled, and the other 50% was replaced with 3x volume of Ringer's lactated solution. Animals were divided into sham and two study groups to receive vehicle (controls) and CY-1503 [corrected] (10 mg/kg intravenously) diluted in 1 mL of normal saline 45 minutes after initiating hemorrhagic shock. The following parameters were analyzed: 7-day survival, liver injury tests, liver tissue myeloperoxidase as an index of neutrophil infiltration, and liver histology. RESULTS: Survival was significantly increased from 48% in the controls to 90% in the CY-1503 [corrected] treated group. Animals treated with the SLe(x) analog showed significantly better mean arterial blood pressure after 15 minutes after resuscitation. Also, the treated group showed a marked decrease in liver enzymes levels at 5 minutes and 4 hours after reperfusion. Neutrophil migration was significantly ameliorated as reflected by decreased myeloperoxidase levels in the SLe(x) analog treated group. Furthermore, we observed improved histologic damage scores in the treated group when compared with controls. CONCLUSIONS: The SLe(x) analog, CY-1503 [corrected], had a protective effect in ischemic livers by decreasing neutrophil migration after hemorrhagic shock and resuscitation. This protective effect also resulted in improved survival and mean arterial blood pressure after resuscitation.

Synthesis and antimuscarinic activity of 2-[N-(ethyl)-(N-beta-hydroxyethyl)]aminoethyl 2,2-diphenylpropionate, a metabolite of aprophen.

The preparation of 2-[N-(ethyl)-(N-beta-hydroxyethyl)]amino-ethyl 2,2-diphenylpropionate (1), a metabolite of aprophen [2-diethylaminoethyl 2,2-diphenylpropionate], is described. Hydrolysis of [2-(2-chloroethyl)ethylamino]ethyl acetate hydrochloride (2) in a basic solution, followed by acidic pH adjustment, gave the ethylcholineaziridinium ion (3) that upon treatment with 2,2-diphenylpropionic acid produced 1 in a 56% yield. Synthetic 1 was found to possess antimuscarinic activities, but was approximately 10-fold less potent than the parent compound aprophen.

Muscarinic receptor subtype specificity of (N,N-dialkylamino)alkyl 2-cyclohexyl-2-phenylpropionates: cylexphenes (cyclohexyl-substituted aprophen analogues).

A series of aprophen [(N,N-diethylamino)ethyl 2,2-diphenylpropionate] analogues, called cylexphenes, were synthesized with alterations in (1) the chain length of the amine portion of the ester, (2) the alkyl groups on the amino alcohol, and (3) a cyclohexyl group replacing one of the phenyl rings. The antimuscarinic activities of these analogues were assessed in two pharmacological assays: the inhibition of acetylcholine-induced contraction of guinea pig ileum, and the blocking of carbachol-stimulated release of alpha-amylase from rat pancreatic acinar cells. These two tissues represent the M3(ileum) and M3(pancreas) muscarinic receptor subtypes. In addition, the analogues were also evaluated for their competitive inhibition of the binding of [3H]NMS to selected cell membranes, each containing only one of the m1, M2, m3, or M4 muscarinic receptor subtypes. The m1 and m3 receptors were stably transfected into A9 L cells. The replacement of one phenyl group of aprophen with a cyclohexyl group increased the selectivity of all the analogues for the pancreatic acinar muscarinic receptor subtype over the ileum subtype by more than 10-fold, with the (N,N-dimethylamino)propyl analogue exhibiting the greatest selectivity for the pancreas receptor subtype, over 30-fold. The cylexphenes also showed a decrease in potency in comparison to the parent compound when examined for the binding of [3H]NMS to the M2 subtype. In agreement with the pharmacological data obtained from the pancreas, the (N,N-dimethylamino)propyl cylexphene 3 demonstrated the greatest selectivity for the m3 subtype, and additionally showed a preference for the m1 and M4 receptor subtypes over the M2 receptor subtype in the binding assay. Thus, this compound showed a potent selectivity according to the pharmacological and binding assays between the muscarinic receptor subtypes of the pancreas and ileum. In both the pharmacological and binding assays, the potency of the analogues decreased markedly when the chain length and the bond distance between the carbonyl oxygen and protonated nitrogen were increased beyond three methylene groups. When the structures of these analogues were analyzed using a molecular modeling program, the bond distance between the carbonyl oxygen and protonated nitrogen was deduced to be more important for the antagonist activity than subtype specificity.

Routine initial computed tomography of the chest in blunt torso trauma.

Computer tomography (CT) is an effective technique in the initial evaluation of the abdomen and head following blunt trauma. To evaluate the role of CT of the thorax, a prospective study comparing routine early thoracic CT scanning with initial chest roentgenogram (CXR) was carried out on 73 patients with blunt torso trauma undergoing concomitant abdominal CT examination. Initial CXR and CT scans were interpreted independently by radiologists in a blinded fashion. CXR diagnosed more bony injuries than CT, while the CT identified pulmonary contusions and effusions more accurately. Only those contusions diagnosed by CXR proved clinically significant. Patient treatment was changed in one case based on CT findings. In the absence of CXR findings, chest CT scanning frequently identifies abnormalities with limited clinical significance. Although more sensitive, CT of the thorax has a limited role in the initial emergent evaluation of victims of blunt torso trauma.

Blood antibodies and uncrossmatched type O blood.

The immediate transfusion of uncrossmatched type O blood in the initial resuscitation of the trauma victim remains controversial. To examine difficulties in crossmatching blood for later transfusions after use of uncrossmatched type O blood, we undertook a prospective 23-month study at a level I trauma center. One hundred thirty-five severely injured patients received uncrossmatched type O blood during the study period. Sixty-one patients (45%) died and 76 patients (56%) underwent emergent operation. There were no major transfusion reactions. Six patients had blood antigen-antibodies present on admission, and such antibodies developed in three patients during hospitalization. We conclude that uncrossmatched type O blood may be used safely in the exsanguinating patient, but blood antigen-antibodies, which may complicate later crossmatching, can develop after its use.

Myocardial contusion without creatine kinase--MB elevation.

Myocardial contusion after chest trauma remains one of the major complexities of trauma care today. Diagnostic methods such as 12-lead electrocardiography and echocardiography, as well as activity of the biochemical marker creatine kinase and the MB subfraction, have not been shown to be sensitive or specific indicators. We report a case of an intraoperatively proved myocardial contusion without creatinine kinase or creatine kinase MB elevation.

Distance geometry of alpha-substituted 2,2-diphenylpropionate antimuscarinics.

Quantitative structure-activity relationships between pharmacological activities and physical properties of a series of 2,2-diphenylpropionate compounds were used to define the topography of the antagonist binding site of muscarinic receptors. XICAMM, a computer molecular modeling program, was used to calculate geometrical and topological values of the compounds. The compounds were tested for their antimuscarinic activities by: (a) inhibition of [N-methyl-3H]scopolamine binding to the muscarinic receptors of N4TG1 neuroblastoma cells, (b) inhibition of carbachol-induced alpha-amylase release from rat pancreas acini, and (c) blocking of acetylcholine-induced contraction of guinea pig ileum. To evaluate as clearly as possible only the effect of the bond distance on the potency of the synthesized antimuscarinics, the compounds contained as many constant features as possible. Neither the hydrophobic nor the ester moieties of the compounds were changed, and the rings containing the protonated nitrogen were saturated and restricted. The antimuscarinic activities obtained from the three assays were significantly correlated with each other, with the exception of two compounds, 9 and 13. The latter two compounds demonstrated specificity for the m3 muscarinic receptor subtype expressed in the pancreas. Furthermore, it was demonstrated that the antimuscarinic activities were significantly related to the bond distances between the carbonyl oxygen (constant electronegative locus) and the protonated nitrogen (center of cationic charge) of the 2,2-diphenylpropionate compounds. Parabolic relationships between the pharmacological activities and bond distances were empirically established. The shortest calculated bond distance of these compounds was approximately 4.4 A, whereas the longest was about 5.9 A. The maximum antimuscarinic potency was observed with a calculated bond distance of about 5.2 A in all three assays.

Injury severity scoring: a comparison of early clinical versus discharge diagnosis.

The Abbreviated Injury Scale (AIS) and the Injury Severity Score (ISS) are objective means of assessing injury. Accepted methodology involves retrospective scoring of injury based on discharge diagnoses. Recently, early clinical scoring, supplemented by review at discharge, has been introduced. A prospective study was instituted to compare these methodologies. Four hundred sixty consecutive victims of blunt trauma were scored using both clinical and retrospective methodologies by independent, blinded observers. Of these, 333 patients had a change in ISS, 174 with a change of greater than four points. The population mean ISS remained unchanged; however, paired values were significantly different (p less than .03). We conclude that either methodology is applicable for studies of large populations of trauma victims. When accurate individual AIS or ISS scoring is required, the clinical method combined with discharge review is most appropriate.

Binary antidotes for organophosphate poisoning: aprophen analogues that are both antimuscarinics and carbamates.

Prophylaxis against organophosphate poisoning can be achieved by pretreatment with physostigmine or pyridostigmine, which are carbamates, and aprophen, which is an anticholinergic agent. Thus, a series of aprophen analogues was synthesized with carbamyl substitutions on the phenyl rings (carbaphens). The rationale behind this design is that such compounds might exhibit most of the therapeutic characteristics of aprophen, as well as the ability to protect prophylactically by chemically masking cholinesterase enzymes. Compounds 4 (dimethylhydroxycarbaphen), 15 (dimethylcarbaphen), and 16 (monomethylcarbaphen) were found to inactivate human butyrylcholinesterase in a time-dependent manner with potencies similar to those of physostigmine or pyridostigmine, and the latter two exhibited almost the same antimuscarinic profile as aprophen. In contrast to the potent inactivation of butyrylcholinesterase by these compounds, marginal inactivation of acetylcholinesterase activity was observed, and only at much higher drug concentrations. The noncarbamylated analogues had no effect on the activity of either cholinesterase. The carbaphen compounds are hence prototype drugs that can interact with either muscarinic receptors or butyrylcholinesterase. Furthermore, these compounds are prodrugs, since after carbamylation of the cholinesterase, the leaving group 14 (hydroxyaprophen) is a potent antimuscarinic itself.

Muscarinic receptor subtype specificity of 5'-(isobutylthio)-adenosine (SIBA) and its analogs.

1. 5'(Isobutylthio)-adenosine (SIBA) and its analogs, at 100 microM, inhibited [3H]N-methyl-scopolamine binding to homogenates of whole brain and cortex (mainly M1 subtype receptors) by 11-30% and to cerebellum (mainly M2 subtype receptors) by 20-39%. 2. At 0.01-1.0 microM, stimulation of [3H]QNB and NMS-inaccessible [3H]QNB binding was observed, with the most induced by 1 microM 3-deaza-SIBA. 3. In contrast, [3H]pirenzepine ([3H]PZ) binding to whole brain and cortex was inhibited in a dose-dependent manner with Ki values in the microM range. 4. As antagonists of acetylcholine-induced contraction of guinea pig ileum (mainly M2 subtype receptors), the analogs were slightly more potent than pirenzepine, but several orders of magnitude less than atropine; the order of potency was opposite that determined for the binding of [3H]PZ to cortex. 5. Thus, SIBA and its analogs may have differential effects on muscarinic receptor subtypes and show some specificity for the M1 receptor subtype.

Pelvic crush injuries with occlusion of the iliac artery.

During a 1-year period, three patients presented with acute traumatic thrombosis of the common or external iliac artery concomitant with a massive crush injury to the pelvis. All had vascular compromise of the involved extremity on initial physical examination, but in two patients with open pelvic trauma, exsanguination, major visceral injury, hypothermia, and a coagulopathy precluded emergency vascular reconstruction. Both required hindquarter amputation for adequate debridement. The third patient presented without exsanguination or visceral trauma. Angiography and vascular reconstruction were undertaken, but myonecrosis compounded the initial vascular compromise, and eventually required a hip disarticulation for debridement. It was concluded that: exsanguination and/or major visceral injury takes priority over emergency vascular reconstruction; soft-tissue injury may preclude limb salvage despite vascular reconstruction. If a cadaveric limb exists, early radical amputation, including hindquarter amputation, should be undertaken.

Use of spiral vein graft to bypass occluded subclavian vein.

A patient presented with massive swelling of left arm secondary to left radio-cephalic fistula and ipsilateral subclavian vein occlusion. This was successfully treated with a spiral vein graft interposed between axillary vein and internal jugular vein.

Desethylaprophen: a metabolite of aprophen with antimuscarinic activities.

The metabolic fate of aprophen hydrochloride (2-diethylaminoethyl 2,2-diphenylpropionate) was studied in rats after intravenous administration. Both 14C-labeled and unlabeled aprophen were used in these studies. Blood samples were collected and analyzed to determine the identities of the metabolites formed. Utilizing high-performance liquid chromatography, desethylaprophen was identified as a major metabolite in ether-extracted samples from rats, and could be detected in blood samples 1 min after intravenous administration. It was most likely formed by N-de-ethylation of aprophen by a cytochrome P-450-dependent monooxygenase. Synthetic desethylaprophen was found to possess cholinolytic activity (i.e., it functioned as a muscarinic antagonist by blocking the contraction of acetylcholine-stimulated guinea pig ileum, the release of alpha-amylase from pancreatic acinar cells stimulated by carbachol, and also by inhibiting the binding of [3H]N-methyl scopolamine to the muscarinic receptors of guinea pig ileum). It was interesting that although the biological effects of desethylaprophen were 100-fold lower than those of aprophen, it was equally able to compete for the binding sites of muscarinic receptors of the guinea pig ileum.

Leishmania braziliensis panamensis: increased infectivity resulting from heat shock.

Promastigotes of Leishmania braziliensis panamensis were subjected to a heat shock transformation yielding an amastigote-like stage. During the process of conversion, the heat-induced differentiating form displayed an increase in infectivity (as determined by lesion size) accompanied by a total protein composition unlike that of the promastigote and a morphology resembling that of the amastigote. These biological/functional changes may be related to an involvement of a heat shock response in the differentiation of leishmania, thus having important implications in the development of prevention and treatment stratagems.

Tubulin synthesis in the regenerating rat superior cervical ganglion: a biphasic response.

Axotomy of a major cranial post ganglionic branch of the superior cervical ganglion of an adult rat resulted in increased radiolabeling of tubulin and microtubule-associated proteins after an in vitro 5-h incubation with [14C]-methionine. The increased incorporation of the radiolabel began at day 1 postoperatively and exhibited a biphasic response, peaking at days 3 and 8.

Studies on the mechanism of cytotoxicity of 3-deazaguanosine in human cancer cells.

The mechanism of toxicity of 3-deazaguanosine was studied in a number of human tumor cell lines by determination of the effects of various purine compounds on the growth of the cells in the presence of the drug and by studies of the effects of 3-deazaguanosine on the metabolism of radiolabeled precursors in these cells. The drug was found to be toxic to all of the cell lines tested. The toxicity was reversible with removal of the drug. None of the purine bases tested could restore normal growth after 48 h exposure to 3-deazaguanosine; the bases were more effective in preventing cytotoxicity when added simultaneously with the drug. Metabolic studies indicated decreased synthesis of DNA, variable inhibition of de novo purine synthesis, and complete inhibition of the enzyme guanosine monophosphate reductase by 3-deazaguanosine.

Effects of ultraviolet light on the in vitro assembly of microtubules.

Exposure of microtubular protein to ultraviolet light inhibits its assembly into morphologically normal microtubules. This effect appeared to result primarily from damage to the tubulin dimers. The damage consisted of a conformational change, a loss of two free sulfhydryl groups, a production of higher molecular weight cross-linked species, and the formation of aggregated amorphous material upon polymerization.

Novel nucleoside inhibitors of guanosine metabolism as antitumor agents.

A detailed study of the inhibition of DR and TR in the SkLu-1 line of human lung adenocarcinoma has shown that TR significantly inhibits this tumor line, probably via inhibition of IMP dehydrogenase by the corresponding TAD analog of NAD. DR exhibited a similar degree of inhibition in this cell line. In a system devised to detect the inhibition of cloning efficiency of the SkLu cells, DR showed a 50% inhibition at 4 X 10(-3) M and TR at 1 X 10(-4) M. When DR and TR were used in combination, the ID50 was decreased to 3 X 10(-5) M. The study of DR in a number of human carcinoma cell lines revealed that de novo purine biosynthesis was significantly inhibited; however, in the SkLu-1 lung carcinoma cells this inhibition was not observed. The synergism observed in this cell line is presently viewed as potentially due to both agents acting on IMP dehydrogenase at different sites.